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Epimedin C, a major flavonoid extracted from Herba Epimedii, is a precursor of minor flavonoid icaritin that is a desired drug candidate with remarkable anti-cancer activities. For enhancing the biotransformation efficiency of icaritin, a novel α- l -rhamnosidase gene was cloned from hyperthermophiles Thermotoga petrophila DSM 13995. TpeRha displayed optimal activity at a pH of 4.5 and a temperature of 90 °C. The Km and Kcat of TpeRha for p-nitrophenyl-α- l -rhamnopyranoside were 2.99 mM and 651.73 s−1, respectively. It displayed broad catalytic ability in cleavage of the outer and inner rhamnopyranosyl moieties on the C-3 carbon of epimedin C. Further, this enzyme was utilized to improve the efficiency of the co-conversion system in transforming epimedin C into icaritin, in combined with a thermostable β-glucosidase Tpebgl1. In addition, a transformation pathway (epimedin C -icariin - icariside I - icaritin) with a high efficiency for icaritin production was screened. After a two-stage transformation under optimized conditions (90 °C, pH 4.5, 80 U/mL of TpeRha and 1.2 U/mL of Tpebgl1), 1 g/L of epimedin C was transformed into 0.4337 g/L of icaritin within 150 min, with a corresponding molar conversion rate of 96.9 %. This is the first report of enzymatic transformation on preparing icaritin from epimedin C by using thermostable glycosidase.

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned ...

A thermostable β-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The β-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable β-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases.

A multimodular hyperthermophilic β‑glucuronidase (TpGUS) from Thermotoga petrophila RKU-1T, belongs to glycoside hydrolase family 2 (GH2), was cloned and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Expression and production of extracellular TpGUS was enhanced through various specific cultivation and induction strategies. Extracellular TpGUS activity was improved by 3.44 and 7 fold in 4 × ZB medium induced with 0.5 mM IPTG and 100 mM lactose, respectively. The enzyme was purified to homogeneity with a single band of 65.6 kDa on SDS-PAGE, using two subsequent steps of anion exchange and hydrophobic interaction chromatography after heat precipitation (70 °C, 1 h). Optimal activity of TpGUS was observed at 95 °C and pH 6.0; and it displayed prodigious thermal stability over a temperature range of 50–85 °C for 12 h at pH 6.0–7.5. Km, Vmax, Vmax Km−1, kcat, and kcat Km−1 were calculated to be 0.7 mM, 227 mmol mg−1 min−1, 324.3 min−1, 164,492.7 s−1 and 234,989.6 mM−1 s−1, respectively using pNPGU as a substrate. Recombinant TpGUS exhibited favorable properties which make this a promising candidate for various biotechnological and pharmacological applications.

In this study, the industrial applications of a highly thermostable α-amylase as a desizer in the textile industry was evaluated. The cloned gene was expressed in different media (ZBM, LB, ZYBM9, and ZB) with IPTG (isopropyl β-D-1-thiogalactopyranoside) used as an inducer. Lactose was also used as an alternate inducer for the T7 promoter system in E. coli. For the large-scale production of the enzyme, different parameters were optimized. The maximum enzyme production was achieved when the volume of medium was 70% of the total volume of fermenter with a 2.0 vvm air supply and 20% dissolved oxygen at a 200 rpm agitation rate. Under all the optimized conditions, the maximum enzyme production was 22.08 U ml−1 min−1 with lactose (200 mM) as an inducer in ZBM medium. The desizing potential of the purified α-amylase enzyme was calculated with different enzyme concentrations (50–300 U ml−1) at different temperatures (50–100 °C), and pHs (4–9) with varying time intervals (30–120 min). The highest desizing activity was found when 150 U ml−1 enzyme units were utilized at 85 °C and at 6.5 pH for 1 h.

Made available in DSpace on 2021-06-25T10:16:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação para o Desenvolvimento da UNESP (FUNDUNESP) It is urgent the transition from a fossil fuel-based economy to a sustainable bioeconomy based on bioconversion technologies using renewable plant biomass feedstocks to produce high chemicals, bioplastics, and biofuels. β-Glucosidases are key enzymes responsible for degrading the plant cell wall polymers, as they cleave glucan-based oligo- and polysaccharides to generate glucose. Monosaccharide-tolerant or -stimulated β-glucosidases have been reported in the past decade. Here, we describe a novel mechanism of β-glucosidase stimulation by glucose and xylose. The glycoside hydrolase 1 family β-glucosidase from Thermotoga petrophila (TpBgl1) displays a typical glucose stimulation mechanism based on an increased Vmax and decreased Km in response to glucose. Through molecular docking and dynamics analyses, we mapped putative monosaccharide binding regions (BRs) on the surface of TpBgl1. Our results indicate that after interaction with glucose or xylose at BR1 site, an adjacent loop region assumes an extended conformation, which increases the entrance to the TpBgl1 active site, improving product formation. Biochemical assays with TpBgl1 BR1 mutants, TpBgl1D49A/Y410A and TpBgl1D49K/Y410H, resulted in decreasing and abolishing monosaccharide stimulation, respectively. These mutations also impaired the BR1 looping extension responsible for monosaccharide stimulation. This study provides a molecular basis for the rational design of β-glucosidases for biotechnological applications. Brazilian Biorenewables National Laboratory (LNBR) Brazilian Center for Research in Energy and Materials (CNPEM) Department of Biochemistry and Tissue Biology Institute of Biology (IB) University of Campinas (UNICAMP) Instituto de Ciências Agrárias (ICA) Universidade Federal de Minas Gerais (UFMG) São Paulo State University (Unesp) Department of Physics Institute of Biosciences Humanities and Exact Sciences Programa de Processos Tecnológicos e Ambientais Universidade de Sorocaba (UNISO) São Paulo State University (Unesp) Department of Physics Institute of Biosciences Humanities and Exact Sciences CNPq: 150664/2013-3 FAPESP: 2010/18198-3 FAPESP: 2011/13242-7 FAPESP: 2012/20549-4 FAPESP: 2015/50590-4 FAPESP: 2016/09950-0 FUNDUNESP: 2532/002/14-PROPe/CDC CNPq: 304816/2017-5 CNPq: 305748/2017-3 CNPq: 428527/2018-3 CNPq: 442352/2014-0

Hot oil reservoirs harbor diverse microbial communities, with many of them inhabiting thermophilic or hyperthermophilic fermentative Thermotogae species. A new Thermotoga sp. strain TFO was isolated from an Californian offshore oil reservoir which is phylogenetically related to thermophilic species T. petrophila RKU-1T and T. naphthophila RKU-10T, isolated from the Kubiki oil reservoir in Japan. The average nucleotide identity and DNA–DNA hybridization measures provide evidence that the novel strain TFO is closely related to T. naphthophila RKU-10T, T. petrophila RKU-1T and can not be differentiated at the species level. In the light of these results, the reclassification of T. naphthophila RKU-10 and strain TFO as heterotypic synonyms of T. petrophila is proposed. A pangenomic survey of closely related species revealed 55 TFO strain-specific proteins, many of which being linked to glycosyltransferases and mobile genetic elements such as recombinases, transposases and prophage, which can contribute to genome evolution and plasticity, promoting bacterial diversification and adaptation to environmental changes. The discovery of a TFO-specific transport system dctPQM, encoding a tripartite ATP-independent periplasmic transporter (TRAP), has to be highlighted. The presence of this TRAP system assumes that it could assist in anaerobic n-alkane degradation by addition of fumarate dicarboxylic acid, suggesting a niche-specific gene pool which correlates with the oil reservoir that T. petrophila TFO inhabits. Finally, T. naphthophila RKU-10, T. petrophila RKU-1T, T. petrophila TFO form a distinct phylogenetic lineage with different geographic origins, share the same type of ecological niche including the burial history of fields. Theses findings might support the indigenous character of this species in oil reservoirs.

International audience; The endo-β-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-β-1,4-mannanase structure determined. The structure exhibits two domains, a (β/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like β-sandwich fold formed of seven β-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.

Mutant homologues of small chemotactic and DNA-binding proteins from thermophilic bacteria Thermotoga petrophila RKU-1 and Thermotoga naphthophila were obtained. These proteins can be expressed in the recombinant form in E. coli cells. A wide range of properties and parameters that are important for isolation of these proteins were revealed: stability in a wide temperature and pH range, high level of expression, solubility, and the possibility of using simple purification schemes with low number of successive steps. The positive effect of proteins on in vitro fibroblasts growth was demonstrated. The described properties of the target proteins indicate the possibility of their use in different biotechnology industries as an inexpensive source of L-amino acids.

In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.V rabote polucheny rekombinantnyĭ khemotaksisnyĭ belok CheW Thermotoga petrophila RKU-1 (TpeCheW) i ego mutantnyĭ gomolog (TpeCheW-mut). Pokazano, chto, nesmotria na nevysokuiu gomologiiu s CheW bakteriĭ gruppy kishechnoĭ palochki, dannye belki ne vyzyvaiut metabolicheskoĭ peregruzki i khorosho ékspressiruiutsia kletkami laboratornykh shtammov E. coli. Vyiavlen shirokiĭ spektr vazhnykh dlia vydeleniia belka TpeCheW-mut svoĭstv i parametrov, takikh kak stabil'nost' v shirokom diapazone temperatur i znacheniĭ rN, vysokiĭ uroven' ékspressii, rastvorimost', a takzhe vozmozhnost' primeneniia prostykh malostadiĭnykh skhem ochistki, vkliuchaia termoobrabotku. Sformulirovany i obosnovany vozmozhnye napravleniia ispol'zovaniia dannogo belka v praktike nauchnykh i prikladnykh issledovaniĭ.

The thermostable GH3 β-glycosidase (Tpebgl3) from Thermotoga petrophila DSM 13995 was immobilized on macroporous resin NKA-9 modified with polyethylenimine (PEI) and glutaraldehyde (named NKA-9II). The properties of NKA-9II were as follows: the optimal conditions were the same as that of the free enzyme (pH 5.0; 90 °C), and the highest activity with cellobiose as the substrate approached 1.7 U/g; the thermostability, pH stability and glucose tolerance were greatly improved; the residual activity of NKA-9II was 68% of the initial activity at the end of 10 repeated cycles. Moreover, it was found that 2 mM Zn2+ increased the relative activity of NKA-9II to 192% and 199% with cellobiose and p-nitrophenyl-β- d -glucopyranoside (pNPG) as substrates, respectively. Meanwhile, Zn2+ could greatly improve the reusability, high-temperature stability, and glucose tolerance of NKA-9II. In particular, 84% of the residual activity of NKA-9II with 2 mM Zn2+ was retained, which was 21% higher than that with free metal ion after incubation at 85 °C for 7 h; when the glucose concentration was 400 mM, the free Tpebgl3 was completely inactivated, and NKA-9II with 2 mM Zn2+ maintained 63% of its initial activity, which was 19.5% higher than the activity of NKA-9II in the absence of Zn2+.

Ginsenoside compound K is an essential ingredient in nutritional supplements, cosmetics, and traditional medicines. However, cultivation for the production of enzymes involved in ginsenoside biotransformation has not been attempted in a fermenter. The host strain Escherichia coli ER2566 and the constitutive pHCE vector were selected for the efficient production of β-D-glycosidase, and expression medium composition to produce Sulfolobus solfataricus β-glycosidase expressed in E. coli was optimized in flask and batch cultures. The total activity of β-Dglycosidase in fed-batch culture using a fermenter increased 14-fold before optimization. S. solfataricus β-D-glycosidase and Thermotoga petrophila α-L-arabinofuranosidase were produced in a fed-batch culture. These two enzymes completely converted protopanaxadiol-type ginsenosides in ginseng leaf extract obtained from discarded ginseng leaves as a renewable substrate to compound K. The effective bioprocess for compound K production developed here will contribute to the industrial biological production of compound K.

One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4polL329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4polL329A and RTX were highly affected by the concentrations of MgCl2 and Mn(OCOCH3)2 as well as those of K4polL329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4polL329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4polL329A and RTX are stable even at 90–100°C, our results suggest that the one-step RT-PCR with K4polL329A or RTX is more advantageous than the current one.

Background Clostridium thermocellum is a cellulolytic anaerobic thermophile that is a promising candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels such as ethanol. It was previously shown that expressing Thermoanaerobacterium saccharolyticum adhA in C. thermocellum increases ethanol yield.In this study, we investigated expression of adhA genes from different organisms in Clostridium thermocellum. Methods Based on sequence identity to T. saccharolyticum adhA, we chose adhA genes from 10 other organisms: Clostridium botulinum, Methanocaldococcus bathoardescens, Thermoanaerobacterium ethanolicus, Thermoanaerobacter mathranii, Thermococcus strain AN1, Thermoanaerobacterium thermosaccharolyticum, Caldicellulosiruptor saccharolyticus, Fervidobacterium nodosum, Marinitoga piezophila, and Thermotoga petrophila. All 11 adhA genes (including T. saccharolyticum adhA) were expressed in C. thermocellum and fermentation end products were analyzed. Results All 11 adhA genes increased C. thermocellum ethanol yield compared to the empty-vector control. C. botulinum and T. ethanolicus adhA genes generated significantly higher ethanol yield than T. saccharolyticum adhA. Conclusion Our results indicated that expressing adhA is an effective method of increasing ethanol yield in wild-type C. thermocellum, and that this appears to be a general property of adhA genes.

Background Cellulose, being the most abundant biopolymer found in nature, can be utilized for bioethanol production to cater the future energy needs. Due to increased usage of fossil fuel it has been predicted that fossil fuel reserves may be depleted by year 2050. These concerns need serious attention and focus should be diverted to renewable fuels that are based on natural resources. Cellulases including exoglucanase (cellobiohydrolases) are the key enzymes that are produced by cellulolytic micro-organisms for the biodegradation of natural resource (cellulose) into fermentable reducing sugars. Many members of genus Clostridium possess supramolecular structures known as cellulosomes which contain various cellulases. Cellulase are composed of catalytic subunits that include endoglucanase, β-glucosidase and cellobiohydrolases which concurrently can catalyse and subsequently convert cellulose into glucose and other sugars. After the action of cellulases, the sugars can be conveniently converted into bioethanol. Objective In the present study, characterization of a thermostable cellobiohydrolase enzyme from Thermotoga petrophila was carried out. The main purpose of this study is the utilization of thermostable cellobiohydrolase along with other cellulases in the process of saccharification of the cellulosic biomass to produce fermentable sugars that could in turn be converted into bioethanol which is the fuel of the future. Method In this article, we propose a framework for achieving our a forementioned object. We started with the cloning of thermophilic cellobiohydrolase gene in mesophilic hosts to ease enzyme production. After cloning of cellobiohydrolase gene, submerged fermentation was performed for intracellular enzyme production. Microbial pellet obtained after centrifugation was sonicated and subjected to ammonium sulphate precipitation. The fraction obtained was purified to isoelectric homogeneity through ion exchange chromatography. Finally SDS analysis of purified cellobiohydrolase was carried out alongwith its characterization, kinetics and thermodynamics studies. Results Purification fold of 4.05 was obtained along with enzyme activity and specific activity of 11.5 U ml-1 min-1 and 66.5 U mg-1, respectively. The molecular mass of purified recombinant enzyme was 37 kDa as calculated by means of SDS-PAGE analysis. The enzyme showed 50% residual activity at 90°C and also at a wide pH range of 4-10. The enzyme retained its activity in the presence of most of the metal ions except Fe+2, Hg+2 and Pb+2. EDTA has an inhibitory effect on the function of the enzyme. The catalytic activity of the enzyme was maintained in the presence of the organic solvents. The enzyme had a Km and Vmax of 4.6 mM and 25.64±1.87 µM min-1 for PNP-β- D-cellobioside under optimal conditions. Conclusion The present study demonstrated that cellobiohydrolase produced from Thermotoga petrophila can be employed in many industries like paper and pulp and food processing. Most recent application of the cellobiohydrolases is their utilization in the production of bioethanol.

A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.

The thermostable β-glucosidase gene from Thermotoga petrophila DSM 13995 was cloned and overexpressed in Escherichia coli. The activity of the recombinant β-glucosidase was 21 U/mL in the LB medium. Recombinant β-glucosidase was purified, and its molecular weight was approximately 81 kDa. The optimal activity was at pH 5.0 and 90 °C, and the thermostability of the enzyme was improved by Ca2+. The β-glucosidase had high selectivity for cleaving the outer and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1, which produced the pharmacologically active minor ginsenoside 20(S)-Rg3. In a reaction at 90 °C and pH 5.0, 10 g/L of ginsenoside Rb1 was transformed into 6.93 g/L of Rg3 within 90 min, with a corresponding molar conversion of 97.9 %, and Rg3 productivity of 4620 mg/L/h. This study is the first report of a GH3-family enzyme that used Ca2+ to improve its thermostability, and it is the first report on the high substrate concentration bioconversion of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 by using thermostable β-glucosidase under high temperature.

Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila . The genes PfBgl1 (GH1 from P. furiosus ), TpBgl1 (GH1 from T. petrophila ) and TpBgl3 (GH3 from T. petrophila ) were cloned and proteins were expressed in Escherichia coli . The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant ( K i = 1100.00 mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides.

Highlights • Lignocresol has great capacity for use in recovery and enzymatic recycling in bioprocesses due to its adsorptive capacity. • The adsorption of TpBgl3 to Lignocresol is higher compared to TpBgl1. • The interactions between lignocresol and enzymes are influenced by electrostatic characteristics, and surface hydrophobicity. • Glucose does not affect the adsorption of enzymes onto lignocresol. • TpBgl1 bound to lignocresol maintains a residual enzymatic activity., Enzymes are essential in many biological processes, including second-generation ethanol production. However, enzymes are one of the main expenses for the industrial process in these days. Several studies have been done to maximize cost savings, however, many processes are still economically infeasible. In this study, we report the synthesis of a suspension of lignocresol for recycling or reuse of enzymes in bioprocesses. In this way, it was performed the adsorption assays between lignocresol and β-glucosidases from Thermotoga petrophila, belonging to the families GH1 and GH3, for the development of a lignocresol-enzyme complex. Our results show that lignocresol maintains greater adsorptive capacity for β-glucosidases than lignin. This capacity can be explained both by its great hydrophobicity and also by electrostatic characteristics. Therefore, all these results demonstrate good adsorption of the enzymes to the lignocresol, demonstrating great potential for enzymatic recycling.

A genomic DNA fragment, encoding a thermotolerant β-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5 kDa protein (by SDS-PAGE) encoded by 1.341 kb gene. The estimated K m and V max values against p-nitrophenyl-β-D-glucopyranoside were 2.8 mM and 42.7 mmol min−1 mg−1, respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.

α-L-arabinofuranosidases (EC 3.2.1.55) participate in the degradation of a variety of L-arabinose-containing polysaccharides and interact synergistically with other hemicellulases in the production of oligosaccharides and bioconversion of lignocellulosic biomass into biofuels. In this work, the structure of a novel thermostable family 51 (GH51) α-L-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was determined at 3.1 A resolution. The TpAraF tertiary structure consists of an (α/β)-barrel catalytic core associated with a C-terminal β-sandwich domain, which is stabilized by hydrophobic contacts. In contrast to other structurally characterized GH51 AraFs, the accessory domain of TpAraF is intimately linked to the active site by a long β-hairpin motif, which modifies the catalytic cavity in shape and volume. Sequence and structural analyses indicate that this motif is unique to Thermotoga AraFs. Small angle X-ray scattering investigation showed that TpAraF assembles as a hexamer in solution and is preserved at the optimum catalytic temperature, 65°C, suggesting functional significance. Crystal packing analysis shows that the biological hexamer encompasses a dimer of trimers and the multiple oligomeric interfaces are predominantly fashioned by polar and electrostatic contacts.

1,3-β-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal β-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.

A hyperthermostable glycoside hydrolase family 51 (GH51) α-L-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-L-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.

Arabinan is a plant structural polysaccharide degraded by two enzymes; a - L -arabinofuranosidase andendo-1,5- a - L -arabinanase. These enzymes are highly diversified in nature, however, little is known abouttheir biochemical and biophysical properties. We have characterized a novel arabinanase (AbnA) isolatedfrom Thermotoga petrophila with unique thermostable properties such as the insignificant decrease ofresidual activity after incubation up to 90 C. We determined the AbnA mode of operation through cap-illary zone electrophoresis, which accumulates arabinotriose and arabinobiose as end products afterhydrolysis of arabinan-containing polysaccharides. Spectroscopic analyses by Far-UV circular dichroismand intrinsic tryptophan fluorescence emission demonstrated that AbnA is folded and formed mainly byb-sheet structural elements. In silico molecular modeling showed that the AbnA structure encompasses afive-bladed b-propeller catalytic core juxtaposed by distorted up-and-down b-barrel domain. The low-resolution structure determined by small angle X-ray scattering indicated that AbnA is monomeric insolution and its molecular shape is in full agreement with the model. 2010 Elsevier Inc. All rights reserved.

Endo-1,4-β-D-mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-β-D-mannanase fromThermotoga petrophilaRKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space groupP212121, with unit-cell parametersa= 58.76,b= 87.99,c= 97.34 Å, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space groupI212121, with unit-cell parametersa= 91.03,b= 89.97,c= 97.89 Å. CryP and CryE diffracted to resolutions of 1.40 and 1.45 Å, respectively.

The endo-1,5-alpha-L-arabinanases belonging to glycoside hydrolase family 43 are of great industrial interest for use in food technology, organic synthesis and biofuel production owing to their ability to catalyze the hydrolysis of alpha-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. In this work, Thermotoga petrophila endo-1,5-alpha-L-arabinanase, a GH43-family member, has been cloned, overexpressed, purified and crystallized. Single crystals were obtained from a solution containing 0.1 M MES buffer pH 6.5, 0.8 M ammonium sulfate, 0.1 M EDTA, 0.1 M L-proline and 5%(v/v) dioxane. X-ray diffraction data were collected to a resolution of 2.86 A using synchrotron radiation and the diffraction pattern was indexed in the tetragonal space group P422, with unit-cell parameters a = b = 83.71, c = 408.25 A.

Made available in DSpace on 2018-12-11T16:41:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 Non-productive adsorption of cellulases onto lignins is an important mechanism that negatively affects the enzymatic hydrolysis of lignocellulose biomass. Here, we examined the non-productive adsorption of two bacterial β-glucosidases (GH1 and GH3) on lignins. The results showed that β-glucosidases can adsorb to lignins through different mechanisms. GH1 β-glucosidase adsorption onto lignins was found to be strongly pH-dependent, suggesting that the adsorption is electrostatically modulated. For GH3 β-glucosidase, the results suggested that the fibronectin type III-like domain interacts with lignins through electrostatic and hydrophobic interactions that can partially, or completely, overcome repulsive electrostatic forces between the catalytic domain and lignins. Finally, the increase of temperature did not result in the increase of β-glucosidases adsorption, probably because there is no significant increase in hydrophobic regions in the β-glucosidases structures. The data provided here can be useful for biotechnological applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. Centro de Ciências Naturais e Humanas (CCNH) Universidade Federal do ABC (UFABC) Departamento de Física e Biofísica Instituto de Biociências UNESP-Univ Estadual Paulista Instituto de Física de São Carlos (IFSC) Universidade de São Paulo (USP) Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) Departamento de Física e Biofísica Instituto de Biociências UNESP-Univ Estadual Paulista

The thermotolerant endo-1,4-β-glucanase gene, of Thermotoga petrophila RKU-1, was cloned and over-expressed in E. coli strain BL21 CodonPlus. Enzyme was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 38 kDa, by purification steps of heat treatment combined with ion-exchange column chromatography. The purified enzyme was optimally active, with specific activity of 530 Umg(-1) against carboxymethyl cellulose (CMC), at pH 6.0 and 95°C and was also stable upto 8 h at 80°C. The enzyme also showed activity against β-glucan barley: 303 %, laminarin: 13.7 %, Whatman filter paper: 0.017 % with no activity against starch and Avicel. The recombinant enzyme exhibited Km, Vmax and Kcat of 12.5 mM, 735 µmol mg-1min-1 and 2351.23 s-1, respectively against CMC as a substrate. The stable recombinant enzyme manifested half life (t1/2) of 6.6 min even at temperature as high as 97°C, with free energy of denaturation (ΔG*D), enthalpy of denaturation (ΔH*D), and entropy of denaturation (ΔS*D) of 98.2 kJ mol(-1), 528.9 kJ mol(-1), and 1.17 kJ mol(-1)K(-1), respectively at 97°C. In addition, the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for hydrolysis of CMC substrate by endo-1,4-β-glucanase were calculated at 95°C as 48.2 kJ mol(-1), 54.6 kJ mol(-1) and -17.4 J mol(-1) K(-1), respectively. The recombinant enzyme saccharified pre-treated wheat straw and bagasse to 3.32 % and 3.2 %, respectively after 6 h incubation at 85°C. Its thermostability, resistance to heavy metal ions and specific activity make this enzyme an interesting candidate for industrial applications.

Made available in DSpace on 2015-10-21T13:14:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-27. Added 1 bitstream(s) on 2015-10-22T09:51:52Z : No. of bitstreams: 1 WOS000350251200050.pdf: 2742659 bytes, checksum: 62233f2378654adf301210ef4f4849a9 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Endo-beta-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65 degrees C when compared to the same enzyme at 20 degrees C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. UNESP Univ Estadual Paulista, Dept Fis, Inst Biociencias Letras &Ciencias Exatas, Sao Jose Do Rio Preto, SP, Brazil Univ Fed ABC UFABC, Ctr Ciencias Nat &Humanas, Santo Andre, SP, Brazil UNESP Univ Estadual Paulista, Inst Biociencias, Dept Fis &Biofis, Botucatu, SP, Brazil Ctr Nacl Pesquisa Energia &Mat, Lab Nacl Ciencia &Tecnol Bioetanol, Campinas, SP, Brazil UNESP Univ Estadual Paulista, Departamento de Física, Inst Biociencias Letras &Ciencias Exatas, Sao Jose Do Rio Preto, SP, Brazil UNESP Univ Estadual Paulista, Inst Biociencias, Departamento de Física, Botucatu, SP, Brazil FAPESP: 2011/13242-7 FAPESP: 2012/21054-9 CNPq: 478900/2012-0 FAPESP: 2008/58037-9 FAPESP: 2012/03503-0 CNPq: 501037/2012-8

The β-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable β-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the β-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two β-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable β-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

Made available in DSpace on 2014-12-03T13:08:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-26Bitstream added on 2014-12-03T13:23:24Z : No. of bitstreams: 1 WOS000333677000097.pdf: 1642455 bytes, checksum: d6caad3a9beac8c58d9f05cc46b29949 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Endo-beta-1,4-mannanase from Thermotoga petrophila (TpMan) is a hyperthermostable enzyme that catalyzes the hydrolysis of beta-1,4-mannoside linkages in various mannan-containing polysaccharides. A recent study reported that TpMan is composed of a GH5 catalytic domain joined by a linker to a carbohydrate-binding domain. However, at this moment, there is no three-dimensional structure determined for TpMan. Little is known about the conformation of the TpMan as well as the role of the length and flexibility of the linker on the spatial arrangement of the constitutive domains. In this study, we report the first structural characterization of the entire TpMan by small-angle X-ray scattering combined with the three-dimensional structures of the individual domains in order to shed light on the low-resolution model, overall dimensions, and flexibility of this modular enzyme at different temperatures. The results are consistent with a linker with a compact structure and that occupies a small volume with respect to its large number of amino acids. Furthermore, at 20 degrees C the results are consistent with a model where TpMan is a molecule composed of three distinct domains and that presents some level of molecular flexibility in solution. Even though the full enzyme has some degree of molecular flexibility, there might be a preferable conformation, which could be described by the rigid-body modeling procedure. Finally, the results indicate that TpMan undergoes a temperature-driven transition between conformational states without a significant disruption of its secondary structure. Our results suggest that the linker can optimize the geometry between the other two domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan. Univ Fed ABC UFABC, Ctr Ciencias Nat & Humanas, Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Fis & Biofis, Sao Paulo, Brazil Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Ciencia & Tecnol Bioetanol, Sao Paulo, Brazil Univ Sao Paulo, Inst Fis, BR-01498 Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Fis & Biofis, Sao Paulo, Brazil CNPq: 2012/21054-9 CNPq: 478900/2012-0 CNPq: 2012/03503-0 CNPq: 501037/2012-8

Orientador: Prof. Dr. Wanius José Garcia da Silva Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2014. A proteina endo-B-1,4-mananase da bacteria Thermotoga petrophila (TpMan) e uma enzima hipertermoestavel que catalisa a hidrolise de ligacoes B-1,4-manosidicas em varios polissacarideos contendo manana. Um recente estudo mostrou que a enzima TpMan e composta de um dominio catalitico GH5 e um dominio de ligacao ao carboidrato CBM27 conectados atraves de um linker, entretanto, ate o momento, a estrutura tridimensional cristalografica nao foi determinada para a enzima completa. Pouco se sabe sobre a conformacao da enzima TpMan intacta como tambem o papel do comprimento e flexibilidade do linker sobre o arranjo espacial dos dominio constitutivos. Neste estudo, nos reportamos a primeira caracterizacao estrutural da enzima TpMan completa utilizando a tecnica de espalhamento de raios-X a baixos angulos (SAXS) combinada com a estrutura tridimensional dos dominios individuais para elucidar o modelo de baixa resolucao, as dimensoes globais, e a flexibilidade desta enzima modular em diferentes temperaturas. Nossos resultados sao consistentes com um linker que apresenta uma estrutura compacta e que ocupa um pequeno volume quando comparado com seu grande numero de residuos de aminoacidos (102 residuos de aminoacidos). Alem disso, a 20 oC os resultados sao consistentes com um modelo onde a enzima TpMan e um monomero composto de tres dominios e que apresenta nivel de flexibilidade molecular em solucao. Mesmo a enzima intacta apresentando algum grau de flexibilidade, existe uma conformacao preferivel, a qual pode ser descrita pelo procedimento de modelagem de corpo rigido. Finalmente, os resultados indicam que a enzima TpMan sofre uma transicao dependente da temperatura entre estados conformacionais de sua estrutura secundaria regular. Nossos resultados sugerem que o linker pode otimizar a geometria entre os outros dois dominios com respeito ao substrato a altas temperaturas. Os estudos apresentados aqui devem proporcionar uma base util para futuros estudos biofisicos da enzima TpMan intacta.

Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1pol(WT)) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4pol(L329A)), in which the mutation of Leu329→Ala is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)(45), the reaction rates of K4pol(L329A) and M1pol(WT) exhibited a saturated profile of the Michaelis-Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)(45) concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)(45) concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases.